| Semen Ziziphi Spinosae(Ziziphus jujuba Mill.var.spinosa(Bunge)hu ex h.F.Chou)is the dry mature seed of the Rhamnaceae plant Ziziphi Spinosae.Mainly produced in Northwest China and the Yellow River Basin.SZS is widely used in TCM clinic and health food development due to its diverse biological activities and significant clinical efficacy.As the first batch of medicinal and edible homologous substances,the development and utilization of SZS resources has always attracted attention.Relevant studies have shown that SZS are rich in protein resources,with a protein content of about 27%.However,there are few studies on SZSP,and there is no in-depth study on its functional characteristics and biological activities,It is of great significance to explore more potential medicinal value and study the biological activity of SZSP systematically.Objective:To optimize the extraction process and study the biological activity of SZSP,reveal the biological activity mechanism of SZSP,discover new plant protein resources with good food characteristics and health care functions,and make full use of SZS medicinal resources.Related health food development provides experimental basis and data support.Methods:1 Optimization of extraction process parameters of SZSP:using protein extraction rate and protein content as evaluation indicators,different designed extraction process routes were screened,and single factor method and response surface method were used to optimize the extraction process parameters of the best extraction process.2 Study on the structure,physicochemical properties and functional properties of SZSP:UV,FTIR,CD and fluorescence spectroscopy were used to analyze the structure and composition of SZSP.The protein molecular weight,amino acid composition,solubility,thermal stability,emulsification,oil and water holding capacity and other physicochemical properties and functional properties were detected to evaluate the food processing properties of SZSP.3 Study on immune activity of SZSP:The immune activity of semen Ziziphi Spinosae protein was evaluated by in vivo and in vitro pharmacological activity experiments.The immunosuppression model of mice induced by CTX was established.The indexes of organ index,immune factor secretion,spleen lymphocyte proliferation,phagocytic capacity of macrophages and NK cell lethality were detected.The spleen,thymus and other immune organs of mice were stained by he and analyzed by immunohistochemistry;Establish LPS-induced RAW 264.7 cell inflammation model,detect cell inflammatory factor secretion,and explore the mechanism of jujube seed protein regulating immune activity based on MAPK and NF-?B signaling pathway.4 Isolation,purification and activity study of SZSP:using ion exchange chromatography and gel chromatography,we studied the isolation and purification of jujube seed protein,and explored the protein isolate of jujube seed protein with significant biological activity.We also used mouse RAW 264.7 macrophages,human cervical cancer cell Hela and human hepatoma cell Hep G2.Objective to evaluate the immunopotentiating and antitumor activities of protein isolated from SZS.5.Optimization of enzymatic hydrolysis process and biological activity of SZSP:the optimal enzymatic hydrolysis process parameters were selected based on the degree of hydrolysis and DPPH free radical scavenging ability;The immune activity of the hydrolysate was evaluated;Based on in vitro antioxidant test and in vivo and in vitro anti fatigue activity test,the biological activity differences of SZSP and SZSPH were compared.Results:1 By comparing different protein extraction methods,the alkali extraction and acid precipitation method is determined as the best extraction method for SZSP,and the parameters of the method are optimized by single factor investigation and response surface method,and the best extraction process is determined as the liquid-to-material ratio.1:20(g/m L),p H=10,extraction temperature 51?,extraction time 49 min,protein extraction rate was 79.71%.The process parameters were tested and verified,and the results showed that the process parameters were accurate and feasible.2 The physicochemical properties and functional properties of SZSP have been systematically studied.The results show that the secondary structure of SZSP is mainly composed of?-sheets.The heat denaturation temperature is 110.5°C.The seed date seed protein is mainly composed of sub-substances with a molecular weight of less than 40 k Da.The 25 and 35 k Da subunits contain glycoprotein structure.SZSP contains an ideal amino acid composition,and the essential amino acid content meets the WHO/FAO requirements for adult intake.SZSP has similar solubility to soybean protein,with oil retention of 2.38 g/g and water retention of 3.63 g/g.The emulsifying and foaming properties first decrease and then increase with the change of p H value.3 Immunomodulatory activity of SZSP was studied based on CTX induced immunosuppression model in mice and LPS induced inflammation model in RAW 264.7 cells.The results showed that SZSP could alleviate the decrease of immune function induced by CTX,protect the growth of immune organs,improve the phagocytic capacity of peritoneal macrophages,and increase the levels of Ig M,Ig A,Ig G and TNF-?And IL-6,and regulate the expression of CD4+and CD8+in immune active organs;SZSP can promote the growth and differentiation of RAW 264.7 cells,stimulate the secretion of NO and inflammatory factors,and is based on MAPK and NF-?B.The mechanism of SZSP regulating the immune activity of RAW 264.7 cell inflammation model was studied by signal pathway.The results showed that SZSP could regulate MAPK and NF-?B by regulating MAPK and NF-?B signaling pathway regulates JNK,ERK and IKB-?Protein expression inhibited LPS induced inflammation in RAW 264.7 cells.4 Sephadex G50 and DEAE-Sepharose Fast Flow chromatography were used to separate and purify two SZSP separation and purification components,S1F2G1 and S1F2G2.The immunological activity of each component was evaluated.The results showed that the S1F2G1 component has a positive effect on RAW 264.7 cells.Good proliferation activity,and can stimulate the secretion of cell inflammatory factors.On this basis,the CCK8 method was used to detect the effect of each component on tumor cell viability,and the results showed that the S1F2G1 component had inhibitory activity on Hela and Hep G2 cells.The inhibitory rates when the dose was 400?g/m L were 71.67%and 54.69%.5 The degree of hydrolysis and DPPH radical scavenging rate were used as evaluation indexes to compare the enzymolysis activities of different proteases.It was determined that alkaline protease was the most suitable enzymolysis protease for SZSP.On this basis,the extraction process parameters were optimized.The optimum enzymolysis conditions were as follows:substrate concentration 5%,enzyme to substrate concentration ratio 2%,enzymolysis temperature 50?,enzymolysis p H=8.0,enzymolysis time 180 min;The results showed that SZSPH had good immunoregulatory activity and could significantly inhibit the production of ROS in RAW 264.7 cells induced by LPS(P<0.01);The antioxidant and anti-fatigue activities of SZSPH showed that,compared with SZSP,the antioxidant activities of SZSPH in superoxide anion,hydroxyl radical,DPPH and ATBS+radical scavenging capacity were significantly improved;The SZSPH could effectively relieve the indexes of exercise fatigue mice,promote the proliferation of C2C12 cells,increase the storage of ATP in C2C12myotubes and the consumption of glucose in culture medium(P<0.01).Conclusion:In this study,the extraction process,structure,physicochemical properties and functional properties of SZSP were studied,and the protein with stable extraction process,clear physicochemical properties and excellent functional properties was obtained;The immunological activity study shows that SZSP can regulate CTX.The expression of CD4+and CD8+in immune organ lymphocytes in the induced mouse immunocompromised model,inhibits the expression of JNK,ERK and IKB?in the MAPK and NF-?B signaling pathways in the RAW 264.7 cell inflammation model induced by LPS,so as to achieve the purpose of regulating immunity;On this basis,for the SZSP with good immunomodulatory activity,the modern protein separation technology and enzymatic hydrolysis technology were used to carry out further separation,purification and enzymatic hydrolysis,and the SZSPH with better immunological activity was obtained.In-depth experimental studies have shown that S1F2G1components have a good inhibitory effect on Hela and Hep G2 tumor cells,suggesting that S1F2G1 components can increase immune activity through the expression of MAPK and NF-?B pathways and achieve anti-tumor effect.SZSPH can exert anti-oxidation and anti-fatigue effects based on effective immune activity.In summary,SZSP is a new and reliable plant protein resource,food,and functional food material with good biological activity. |
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